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Division of Clinical and Molecular Endocrinology, University Hospitals of Cleveland, and Case Western Reserve University, Cleveland, Ohio 44106
Address all correspondence and requests for reprints to: Baha M. Arafah, M.D., Division of Clinical and Molecular Endocrinology, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: bxa{at}po.cwru.edu.
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Abstract |
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Patients were characterized to have normal HPA (n = 47) or abnormal HPA (ABN-HPA, n = 35) function based on their respective responses to insulin-induced hypoglycemia. Patients also underwent low-dose Cortrosyn (1 µg, LDC) and standard-dose Cortrosyn stimulation testing.
All patients with ABN-HPA had very low age- and gender-matched serum DHEA-S levels. When the normal response to LDC was set at a cortisol level of at least 18.1 µg/dl, 10 of 31 patients with ABN-HPA exhibited normal responses. Receiver operating characteristic curves for baseline DHEA-S and for maximal cortisol responses to LDC had areas of 0.984 (confidence interval, 0.962–1.000) and 0.893 (confidence interval, 0.817–0.969), respectively.
LDC- or SDC-stimulated serum cortisol levels have significant limitations in defining HPA function. A normal age- and gender-specific serum DHEA-S level makes the diagnosis of corticotropin deficiency extremely unlikely. However, when serum DHEA-S levels are low, further testing is necessary to define HPA function.
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Introduction |
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The diagnosis of central adrenal insufficiency continues to be difficult, particularly when the deficiency is partial. Though insulin-induced hypoglycemia (IIH) is considered to be the gold standard test, it is commonly associated with discomfort, requires close monitoring, and has limited use in children, the elderly, and patients with heart disease (1, 2). The standard-dose Cortrosyn (SDC) test was introduced as an alternative test for establishing the diagnosis of primary (and subsequently, that of central) adrenal insufficiency. However, several reports demonstrating normal cortisol responses to the SDC, in patients with documented central adrenal insufficiency, have raised concerns about the test’s validity in this condition (3, 4). This led to the introduction of the low-dose Cortrosyn test (LDC), where 1 µg of Cortrosyn (instead of 250 µg) is given as the test dose (4, 5, 6, 7, 8). The latter dose of Cortrosyn mimics more closely the maximum endogenous corticotropin stress response generated by the activation of the hypothalamic-pituitary unit. The sensitivity and specificity of the LDC test depend on the cortisol cutoff value used to define a normal response (5, 6), although they remain less than that of IIH in establishing the diagnosis of partial central adrenal insufficiency (1, 2, 6, 7, 8).
In the present investigation, we sought an alternative approach to assess the integrity of the HPA axis. Dehydroepiandrosterone (DHEA) and its sulfated ester (DHEA-S) are adrenal androgen precursors secreted by the zona reticularis, under the dominant regulation of corticotropin (9). Serum levels of DHEA-S are affected by several factors, the most important of which are age, gender, chronic illness, and the prior use of glucocorticoids (9). Earlier reports indicate that, in treated hypopituitary patients, serum DHEA-S levels are uniformly very low (10, 11, 12, 13). However, data on the use of DHEA-S in the assessment of adrenal function are limited.
We have conducted a prospective study to investigate the value of serum DHEA-S measurements in assessing HPA function. The study involved patients at risk for having central adrenal insufficiency; namely those with large pituitary tumors. We postulated that a normal DHEA-S serum level is a reliable indicator of an intact HPA axis. The study included patients with pituitary tumors who had never previously received glucocorticoids, as well as normal healthy volunteers. Our data are consistent with the hypothesis that a normal DHEA-S serum level makes the diagnosis of central adrenal insufficiency extremely unlikely. However, because serum DHEA-S levels can be lowered by other factors, a low level does not necessarily indicate impaired adrenal function.
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Subjects and Methods |
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Consecutive adult patients with newly diagnosed large (>1.5 cm) pituitary adenomas were included in the study. All patients gave informed consent and underwent testing of pituitary function as previously described (14, 15). Only data pertinent to the assessment of HPA axis function are presented here. Patients with Cushing’s disease were excluded. None of the patients had received glucocorticoids, and none of the women were receiving estrogen therapy. Tumors were classified on the basis of the dominant cell type on immunohistochemical staining of multiple sections of the resected adenoma. Patients with prolactin (PRL)-secreting adenomas who did not have surgery were classified as such on the basis of very high (>300 µg/liter) serum PRL levels.
The study population comprised the following three groups: 1) Group I consisted of 35 patients who had abnormally low HPA (ABN-HPA) function as defined by either a serum cortisol response of less than 18.5 µg/dl (510 nM) during IIH and/or a morning cortisol less than 3 µg/dl (83 nM). 2) Group II consisted of 47 patients with pituitary macroadenomas, whose HPA function was documented to be normal (NL-HPA), as defined by a serum cortisol level of more than 18.5 µg/dl (510 nM) in the basal state or in response to IIH. 3) Group III consisted of 21 healthy volunteers, comparable in age and gender with the two groups.
After testing, patients with ABN-HPA were started on hydrocortisone replacement therapy that was withdrawn immediately after surgical removal of the tumor as described previously (14, 15). Patients with NL-HPA underwent surgical removal of the adenoma without any glucocorticoid therapy before, during, or after the procedure as described previously (14, 15). The safety of this approach has been demonstrated repeatedly since the original report was published (16). After surgery, patients with normal preoperative HPA function are closely monitored, and normal adrenal function is documented before discharge.
Study protocol
Testing was performed preoperatively, in an ambulatory setting, on different days, in a random order for the same subject, mostly in the morning. Healthy volunteers were tested mostly in the early afternoon.
IIH. Variable doses of insulin (0.05–0.3 U/kg) were injected iv to induce hypoglycemia (glucose < 40 mg/dl or 2.22 mM). Plasma corticotropin and cortisol measurements were obtained before and every 15 min, for 120 min, as described previously (12, 13). The highest serum cortisol level during the test was considered the maximal adrenal response. The test was performed in both groups of patients with pituitary tumors. A cortisol response of more than 18.5 µg/dl (510 nM) was considered normal (15, 17).
SDC. Serum cortisol and DHEA-S levels were measured before and 30, 45, and 60 min after the iv injection of 250 µg Cortrosyn.
LDC. One milliliter of normal saline was drawn from a 250-ml bag, mixed with 250 µg Cortrosyn, and reinjected into the 249-ml bag. After mixing, 1 ml (equivalent to 1 µg) was drawn from the bag and injected iv into patients of groups I and II. Measurements were obtained in a manner similar to that of the SDC.
We assessed the sensitivity of each test at two different cutoff levels of cortisol that are frequently reported in the literature (>18.1 and 19.9 µg/dl, corresponding to 500 and 550 nM, respectively), as indicative of adequate adrenal function for either LDC or SDC (6, 7, 8, 9).
Laboratory methods
Serum DHEA-S levels were measured by RIA, using a Coat-A-Count kit (Diagnostic Products Corp. DPC, Los Angeles, CA). The lower limit of detectability was 1.1 µg/dl, and the inter- and the intraassay coefficients of variation were 9.7% and 4.4%, respectively. Serum cortisol levels were measured by immunoassay using a chemiluminescence technique (Centaur/Bayer Diagnostics, Tarrytown, NY). The lower limit of detectability was 0.2 µg/dl, and the inter- and the intraassay coefficients of variation were 8.9% and 4.3%, respectively.
Statistical analysis
Baseline demographic and clinical variables were compared between groups, using Kruskal-Wallis followed by Mann-Whitney U tests for continuous outcomes and 
2/exact Fisher tests for categorical outcomes. Statistical differences in serum cortisol and DHEA-S levels among all groups were determined using Kruskal-Wallis, followed by Mann-Whitney U test in between two groups. In addition, receiver operating characteristic (ROC) curves were plotted for both cortisol and DHEA-S serum levels, using IIH as the gold standard to distinguish between NL-HPA and ABN-HPA groups. Because serum DHEA-S levels are age- and gender-dependent, the respective values in the groups were further analyzed according to subgroups determined by gender and age intervals of 20 yr. The data are presented as mean ± SD unless stated otherwise.
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Results |
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), mean age was higher in group I patients when compared with group II but not when compared with the volunteers. Tumor type distribution was different for gonadotrophs and GH-secreting tumors but not for prolactinomas or nonsecreting adenomas. All patients in group I had hypoadrenalism (by definition) and hypogonadism, and 25 of 35 had hypothyroidism. In contrast, only 28 of 47 and five of 47 patients of group II had hypogonadism and hypothyroidism, respectively.
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Maximal serum cortisol levels during IIH were higher (P < 0.001) in patients with NL-HPA (26.9 ± 6.9 µg/dl, or 742.2 ± 190.4 nM) than in those with ABN-HPA (10.3 ± 6.1 µg/dl, or 284.2 ± 168.3 nM). As shown in Table 2, patients with ABN-HPA had lower mean basal and Cortrosyn-stimulated (SDC and LDC) serum cortisol concentrations, compared with the respective values in those with NL-HPA. Maximal serum cortisol responses to SDC in patients with NL-HPA were similar to those of healthy volunteers. Baseline serum cortisol levels in healthy volunteers were lower than those of patients with NL-HPA, likely because the former group was tested in the early afternoon (Table 2). Although baseline serum cortisol is known to vary with time of the day, the Cortrosyn-stimulated levels do not vary with changes in the time of testing (17).
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. Using two different cutoff values to define normal serum cortisol response to either the LDC or SDC tests (18.1 or 19.9 µg/dl corresponding to 500 and 550 nM, respectively), the number of individual responses within each group is presented in Table 3. The data indicate that, even when one uses a higher cutoff value of 19.9 µg/dl (550 nM) for either the SDC or LDC test, a significant portion of patients (41% and 10%, respectively) with documented central adrenal insufficiency, as defined herein, would have been incorrectly characterized as having NL-HPA.
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Patients with ABN-HPA had drastically reduced basal and stimulated serum DHEA-S concentrations (Table 2
). Patients with NL-HPA function had mean basal and stimulated serum DHEA-S levels that were similar to those of healthy volunteers (Table 2). Individual and age- and gender-matched serum DHEA-S levels are presented in Fig. 2, in relation to the 5th percentile of our laboratory reference data. Whereas all 33 patients with ABN-HPA had clearly low age- and gender-matched serum DHEA-S concentrations, nine of 47 patients with NL-HPA had levels below the lower limits of their respective normal ranges (Table 4). Whereas two of those values were only marginally decreased, the remaining seven values were moderately low (Fig. 2).
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), all of whom had low gender- and age-matched serum DHEA-S levels. Similarly, there were four of 39 patients with NL-HPA whose LDC stimulated serum cortisol levels were no more than 18.1 µg/dl (500 nM), and all had normal serum DHEA-S concentrations. The impact of gonadal dysfunction on serum DHEA-S levels was examined in the group of patients with NL-HPA. The data indicate that six of 27 patients with hypogonadism, compared with three of 20 with normal gonadal function, had low serum DHEA-S levels (P = 0.605). As a group (n = 47), serum DHEA-S levels in hypogonadal patients (120 ± 40 µg/dl, or 3.26 ± 1.1 µM, n = 27) were similar to those with normal gonadal function (133 ± 55 µg/dl, or 3.61 ± 1.49 µM, n = 20). Similar findings were noted when the data on the 18 men with NL-HPA were examined separately. Specifically, four of 12 men with hypogonadism and two of six men with normal gonadal function had low serum DHEA-S levels (P = 1.00). Mean serum DHEA-S levels in the latter two subgroups of men were similar (121.8 ± 54 µg/dl, or 3.31 ± 1.47 µM vs. 115.6 ± 66 µg/dl, or 3.14 ± 1.79 µM, respectively).
ROC curve for DHEA-S and cortisol
Using results from LDC, maximal cortisol and baseline DHEA-S levels were plotted in an ROC curve (Fig. 3). The area under the curve for maximal cortisol was 0.893 [confidence interval (CI), 0.817–0.969], reaching 100% sensitivity when the cortisol level is above 24.4 µg/dl, and 100% specificity when the level is less than 16.9 µg/dl, as defined by IIH. Using DHEA-S values, even unadjusted for age and gender, the area under the curve was 0.984 (CI, 0.962–1.000), with near-100% sensitivity when the absolute DHEA-S value is above 53.5 µg/dl, and 100% specificity when the value is less than 14.0 µg/dl, as defined by IIH (Fig. 3). The area under the curve for the serum DHEA-S levels was significantly different (P = 0.031) from that of LDC-stimulated serum cortisol concentrations. Similarly, the baseline DHEA-S and maximal cortisol, using the SDC test, were plotted for all three groups. The area under the curve for cortisol was 0.894 (CI, 0.820–0.968) and for DHEA-S was 0.986 (CI, 0.969–1.000).
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Discussion |
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Physiologically, the use of Cortrosyn stimulation in establishing the diagnosis of central adrenal insufficiency was based on the assumption that corticotropin acts as a trophic factor for maintenance of the adrenal cortex (1). Without corticotropin, the adrenal cortex atrophies and would not be expected to respond promptly to exogenously administered Cortrosyn. However, because corticotropin deficiency is most often partial rather than complete, some patients may respond well to exogenous Cortrosyn, even though their corticotropin secretion is subnormal. The normal cortisol response seen with the high-dose Cortrosyn stimulation test in 50% of adrenally insufficient patients is, in part, caused by the use of supraphysiologic doses of corticotropin. Peak plasma corticotropin levels, after iv administration of 250 µg Cortrosyn (i.e. SDC), are reported to reach 60 µg/liter, or 13.2 nM (17). In contrast, plasma corticotropin levels reach approximately 100 ng/liter (22 pM during the IIH. It is for this reason that the LDC test is less likely to give falsely normal values than the SDC test in patients with documented central adrenal insufficiency. However, our data demonstrate that, even when the dose of Cortrosyn was lowered with the use of LDC, some patients, albeit fewer in number, continue to have normal Cortrosyn-stimulated levels despite being adrenally insufficient.
It is unclear as to why DHEA-S values would be that low even when corticotropin deficiency was only partial. It is suggested that, in cases of early corticotropin deficiency, the adrenal would preferentially divert more of its resources toward cortisol production. Such instances of cortisol/DHEA-S dissociation have been described in a variety of clinical situations, even when corticotropin levels are abundant (9). Mechanisms regulating this dissociation are not well defined but may involve factors such as preferential zoning of blood flow away from the reticularis and toward the fasiculata layer and perhaps altered adrenal enzyme activity.
DHEA-S, the most abundant steroid in the circulation, is practically all secreted by the adrenal glands, with minor contribution by the testis (18, 19). DHEA-S is fully derived from the sulfation of DHEA, and levels of both of these steroids correlate under most clinical circumstances. In longitudinal studies, levels of DHEA-S have shown tracking in the same individual (19). Furthermore, DHEA-S has a half-life of 10–20 h and does not follow a circadian rhythm. Basal levels correlate closely and are approximately 10–15% lower than Cortrosyn-stimulated levels. All of these factors make a single measurement of DHEA-S practical and reliable.
We do not believe that the concomitant pituitary hormone abnormalities in some of the patients had a significant impact on our findings or conclusions. Our data indicate that serum DHEA-S levels in men and women with NL-HPA function were not affected by their respective gonadal function. Published studies by other investigators are supportive of this view (19, 20, 21). Even though some of the women in our study were menopausal, it is unlikely that DHEA-S data were compromised. In a prospective study of 172 women in whom DHEA-S levels were followed over a period of 7 yr, through the menopause, there was no correlation between DHEA-S and biochemical menopausal changes (20). Likewise, DHEA-S levels seem independent of states of gonadal dysfunction (21). PRL is reported to increase serum DHEA-S by mechanisms that are unclear but may be attributable to increased adrenal enzyme expression and/or decreased DHEA-S clearance (22). This was not found to be a significant contributor in the presence of corticotropin deficiency (10). Furthermore, the number of patients with PRL-secreting tumors in the two groups, studied herein, was similar.
One limitation of the present study is the relatively small sample size, especially when the population is divided into subgroups. Nevertheless, the uniformity of the response supports our hypothesis that normal DHEA-S levels indicate an intact HPA function. Because serum DHEA-S levels normally decline progressively with advancing age, a low serum level in the elderly population could be difficult to interpret. Based on our study and the ROC curve generated from our data, we believe that the serum DHEA-S level that gives 100% sensitivity should be considered as the minimum required to define normal, in any group or gender. We believe that, in addition to being in the age- and gender-matched range, serum DHEA-S levels have to be more than 54.5 µg/dl (1.48 µM) to be considered normal.
In view of the cortisol/adrenal androgen dissociation discussed above and the fact that multiple factors such as age, gender, and previous steroid use modulate DHEA-S production, one should judge the clinical significance of low serum DHEA-S levels with caution. One major group of patients in whom interpretation of low serum DHEA-S levels would not be reliable are those who received exogenous glucocorticoids during the year before testing and those who have chronic illnesses. In the former group of patients, DHEA-S levels can remain low for some time despite normal cortisol secretion. Thus, even though partial or complete deficiency of corticotropin results in a low serum DHEA-S level, it would be imprudent to assume that the converse is always true. Specifically, because many factors (such as advancing age, chronic illnesses, and prior and current use of glucocorticoids) can independently result in low serum DHEA-S levels, one should interpret such low levels with clinical scrutiny. One should keep the clinical context of the patient in focus in the evaluation of low serum DHEA-S levels.
Our data clearly demonstrate that, when serum DHEA-S levels are normal, the diagnosis of corticotropin deficiency is extremely unlikely. Although our study included only patients with pituitary adenomas, there is no apparent reason to limit its applicability to such patients. We believe that the conclusions and recommendations can be extended to other patients investigated for the possibility of central adrenal insufficiency. In view of our findings, we believe that the biochemical assessment of adrenal function should include measurements of cortisol and DHEA-S serum levels. In the absence of corticosteroid-binding globulin excess (e.g. with estrogen therapy), a random unstimulated serum cortisol level of more than 18.5 µg/dl (510 nM) and/or a normal age-and gender-adjusted serum DHEA-S level would practically and effectively rule out the diagnosis of adrenal insufficiency. As stated herein, serum DHEA-S levels are extremely helpful only when they are normal. Thus, when they are low, assessment of adrenal function should rely on clinical evaluation and measurements of serum cortisol level and its response to stimulation. The LDC provides a simple, noninvasive, though imperfect test. An LDC-stimulated serum cortisol of less than 16.9 µg/dl (466 nM) confirms the diagnosis of corticotropin deficiency, whereas a level of more than 24.4 µg/dl (673 nM) rules it out. When the LDC-stimulated serum cortisol levels are intermediate, further testing would be necessary, as clinically indicated. It is in those instances where the value of serum DHEA-S is most valuable. It is important to point out that DHEA is currently available as an over-the-counter therapeutic agent or a supplement and could potentially be used by some patients being investigated for the possibility of adrenal insufficiency. Thus, it would be prudent to entertain the latter possibility as a pitfall in the interpretation of a normal random serum DHEA-S measurement.
In summary, a normal DHEA-S level makes the diagnosis of corticotropin deficiency extremely unlikely, especially when the Cortrosyn stimulation test is normal. We recommend measurements of serum DHEA-S as an important component in the assessment of HPA function. The data should be interpreted with age, gender, and other factors modulating serum DHEA-S levels in mind.
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Acknowledgments |
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Footnotes |
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A preliminary report of the data was presented at the 83rd Annual Meeting of The Endocrine Society, Denver, CO, 2001.
Abbreviations: ABN-HPA, Abnormal HPA; CI, confidence interval; DHEA, dehydroepiandrosterone; DHEA-S, DHEA sulfate; HPA, hypothalamic-pituitary-adrenal; IIH, insulin-induced hypoglycemia; LDC, low-dose Cortrosyn; NL-HPA, normal HPA; PRL, prolactin; ROC, receiver operating characteristic; SDC, standard-dose Cortrosyn.
Received March 13, 2003.
Accepted August 6, 2003.
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